Abstract

Cognitive dysfunction is an early clinical hallmark of Huntington's disease (HD) preceding the appearance of motor symptoms by several years. Neuronal dysfunction and altered corticostriatal connectivity have been postulated to be fundamental to explain these early disturbances. However, no treatments to attenuate cognitive changes have been successful: the reason may rely on the idea that the temporal sequence of pathological changes is as critical as the changes per se when new therapies are in development. To this aim, it becomes critical to use HD mouse models in which cognitive impairments appear prior to motor symptoms. In this study, we demonstrate procedural memory and motor learning deficits in two different HD mice and at ages preceding motor disturbances. These impairments are associated with altered corticostriatal long-term potentiation (LTP) and specific reduction of dendritic spine density and postsynaptic density (PSD)-95 and spinophilin-positive clusters in the cortex of HD mice. As a potential mechanism, we described an early decrease of Kalirin-7 (Kal7), a guanine-nucleotide exchange factor for Rho-like small GTPases critical to maintain excitatory synapse, in the cortex of HD mice. Supporting a role for Kal7 in HD synaptic deficits, exogenous expression of Kal7 restores the reduction of excitatory synapses in HD cortical cultures. Altogether, our results suggest that cortical dysfunction precedes striatal disturbances in HD and underlie early corticostriatal LTP and cognitive defects. Moreover, we identified diminished Kal7 as a key contributor to HD cortical alterations, placing Kal7 as a molecular target for future therapies aimed to restore corticostriatal function in HD.

Spinophilin-immunoreactive puncta are reduced in the motor cortex but not in the striatum…

Figure 5.

Spinophilin-immunoreactive puncta are reduced in the motor cortex but not in the striatum of HdhQ7/Q111 mice. Representative confocal images showing spinophilin (red) positive clusters in the motor cortex (A and B) and dorsal striatum (C and D) of WT HdhQ7/Q7 and knock-in HdhQ7/Q111 mice at 2 (A and C) and 8 (B and D) months of age. Spinophilin-immunoreactive puncta were counted and mean size evaluated in layers I, II/III and V of motor cortex area 1 (M1) and in the DL and DM striatum. Quantitative analysis is shown as mean ± SEM (n = 5–6 animals per group). A specific reduction in spinophilin-immunoreactive puncta was found in the cortex but not in the striatum of knock-in HdhQ7/Q111 mice from early disease stages. Statistical analysis was performed using Student's two-tailed t test. *P < 0.05; **P < 0.01 compared with HdhQ7/Q7 mice. Scale bar, 5 µm.

Figure 6.

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PSD-95-immunoreactive puncta are decreased at…

Figure 6.

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PSD-95-immunoreactive puncta are decreased at early disease stages in the motor cortex of…

Figure 6.

PSD-95-immunoreactive puncta are decreased at early disease stages in the motor cortex of HdhQ7/Q111 mice. Representative confocal images showing PSD-95 (green) positive clusters in the motor cortex (A and B) and dorsal striatum (C and D) of WT HdhQ7/Q7 and mutant knock-in HdhQ7/Q111 mice at 2 (A and C) and 8 (B and D) months of age. Cortical PSD-95-immunoreactive puncta were counted and mean size evaluated in layers I, II/III and V of motor cortex area 1 (M1) and in the DL and DM striatum. Quantitative analysis is shown as mean ± SEM (n = 5–6 animals per group). A specific reduction in PSD-95-immunoreactive puncta was found in cortex but not striatum of HdhQ7/Q111 mice at early disease stages. At more advanced disease stages, a reduction in PSD-95-immunoreactive puncta was also found in the dorsal striatum. Statistical analysis was performed using Student's two-tailed t test. *P < 0.05; **P < 0.01; ***P < 0.001 compared with HdhQ7/Q7 mice. Scale bar, 5 µm.

Figure 7.

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Levels of Kal7 are reduced…

Figure 7.

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Levels of Kal7 are reduced in the cortex but not in the striatum…

Figure 7.

Levels of Kal7 are reduced in the cortex but not in the striatum of HdhQ7/Q111 mice at early disease stages. Representative western blots showing the indicated subunits of glutamate receptors, pre- and postsynaptic scaffolding proteins and protein kinases in the total cortical (A) and striatal (B) extracts from 2- and 8-month-old WT HdhQ7/Q7 and knock-in HdhQ7/Q111 mutant mice. α-Tubulin was used as a loading control. A specific reduction in Kal7 was found in the cortex but not in the striatum of HdhQ7/Q111 mutant mice compared with HdhQ7/Q7 WT mice at 2 months of age. At more advanced disease stages, a reduction in other synaptic-related proteins was found. Histograms represent the mean ± SEM and are expressed as percentage of WT animals (n = 5–8 animals per genotype). Statistical analysis was performed using Student's two-tailed t test. *P < 0.05; **P < 0.01 compared with HdhQ7/Q7 mice.

Figure 8.

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Kal7 levels are reduced in…

Figure 8.

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Kal7 levels are reduced in the cortex of R6/1 mice and in the…

Figure 8.

Kal7 levels are reduced in the cortex of R6/1 mice and in the cortex and putamen of HD brains. (A) Representative western blots showing Kal7 and α-tubulin as a loading control in total cortical and striatal extracts from 2- and 3-month-old WT and R6/1 mice. Histograms represent the mean ± SEM (n = 5–8 animals per genotype). Statistical analysis was performed using Student's two-tailed t test. **P < 0.01; ***P < 0.001 compared with HdhQ7/Q7 mice. (B) Representative western blots showing Kal7, GluN1, GluA1 and α-tubulin as a loading control in total cortical and putamen extracts from control (n = 4–7) and HD samples (n = 5–7). Histograms represent mean ± SEM and are expressed as percentage of control samples. Student's two-tailed t test was performed. *P < 0.05; **P < 0.01; ***P < 0.001 compared with control human samples.

Figure 9.

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Rac1 activity is reduced in…

Figure 9.

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Rac1 activity is reduced in the cortex of Hdh Q7/Q111 mice. Representative western…

Figure 9.

Rac1 activity is reduced in the cortex of HdhQ7/Q111 mice. Representative western blot of cortical extracts isolated from WT HdhQ7/Q7 and mutant HdhQ7/Q111 mice at 8 months of age showing Rac1-GTP and total Rac1 levels. Activated (GTP-bound) Rac1 was detected by immunoblotting of pull-down experiments from cortical extracts. Diminished Rac1 activity was found in HdhQ7/Q111 mutant mice compared with HdhQ7/Q7 WT mice (n = 5 animals per genotype), whereas similar total Rac1 levels were found between genotypes. Histograms represent mean ± SEM and are expressed as percentage of WT animals. Student's two-tailed t test was performed. *P < 0.05 compared with HdhQ7/Q7 mice.

Figure 10.

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Reduced levels of Kal7 associates…

Figure 10.

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Reduced levels of Kal7 associates with decreased number of synapse in mature cortical…